Protein ubiquitination is a widespread reversible post-translational modification that is used by eukaryotic cells as a major regulatory mechanism to alter protein stability, localization, conformation and activity of the modified substrates. Ubiquitin is a highly conserved 76 amino acid protein and conjugation of ubiquitin to target proteins requires a series of enzymatic reactions (Pickart, 2001): After activation of ubiquitin through the formation of a thioester bond between its C-terminal glycine and the active site cysteine of the ubiquitin activating protein E1, it is transferred by subsequent trans-thiolation reactions to a cysteine residue on a ubiquitin conjugating enzyme, E2. Finally, a substrate specifity determining ubiquitin ligase (E3) transfers ubiquitin bound to E2 to a specific polypeptide target, forming an isopeptide bond between the C-terminal glycine of ubiquitin and the ε-amino group of a lysine present in the target. Ubiquitin itself can also undergo ubiquitination on any of its seven lysine residues resulting in formation of distinct poly-ubiquitin chains (Komander, 2009). Depending on the number and type of ubiquitin moieties attached, a protein can be mono-ubiquitinated, multiple mono-ubiquitinated at different lysines and poly-ubiquitinated. This heterogeneity plays a critical role in molecular recognition of the modified proteins by numerous types of ubiquitin binding proteins (Hicke, 2005). In addition, the size and type of ubiquitin conjugates serve as a specific code for determination of the fate of tagged proteins. For example, modification by Lysine-48 poly-ubiquitin chains directs proteins mainly for degradation by the 26S proteasome, whereas mono-ubiquitination and Lysine-63 type poly-ubiquitination have been associated with regulation of several cellular processes including signal transduction, endocytosis, chromatin rearrangement and DNA repair (Haglund, 2005; Mailand, 2007; Messick, 2009; Pandita, 2009).
Due to the large dynamic range of protein expression and a generally low stoichiometry of ubiquitination on proteins, it is challenging to identify the ubiquitinated proteins and ubiquitination sites on these proteins. As a consequence of these challenges there are only a limited number of reports investigating protein ubiquitination on a proteomic scale. Mass spectrometric approaches have mainly been limited to affinity enrichment of ubiquitinated proteins using overexpressed tagged ubiquitin (Gururaja, 2003; Peng, 2003; Danielsen, 2011; Argenzio, 2011) or poorly-working antibodies raised against ubiquitin (Argenzio, 2011; Matsumoto, 2005; Vasilescu, 2005). Another strategy to enrich directly modified peptides from ubiquitinated targets using antibodies against the remnant diglycine of ubiquitin on lysine residues after tryptic digestion has been recently developed.
Because of the complex nature of ubiquitination, its heterogeneity and the lack of suitable procedures there is a need for new strategies to study ubiquitination on a proteomic scale.
Ubiquitin has been implicated in a number of cellular processes including: signal transduction, cell-cycle progression, receptor-mediated endocytosis, transcription, organelle biogenesis, spermatogenesis, response to cell stress, DNA repair, differentiation, programmed cell death, and immune responses (e.g., inflammation). Ubiquitin also has been implicated in the biogenesis of ribosomes, nucleosomes, peroxisomes and myofibrils. Thus, ubiquitin can function both as signal for polypeptide degradation and as a chaperone for promoting the formation of organelles.
Deregulation of ubiquitination has been implicated in the pathogenesis of many different diseases. For example, abnormal accumulations of ubiquitinated species are found in patients with neurodegenerative diseases such as Alzheimer's as well as in patients with cell proliferative diseases, such as cancer.
While the importance of its biological role is well appreciated, the ubiquitin pathway is inherently difficult to study. Generally, studies of ubiquitination have focused on particular polypeptides. For example, site-directed mutagenesis has been used to evaluate critical amino acids which form the “destruction boxes” or “D-boxes” of cyclin, sites which are rapidly poly-ubiquitinated when cyclin is triggered for degradation. Moreover, the ubiquitin-proteosome system is the principal mechanism for the turnover of short-lived polypeptides, including regulatory polypeptides.
Although mass spectrometry offers a powerful tool for identifying ubiquitin substrates, a number of unresolved issues remain. Despite many advances, MS data is inherently biased towards more abundant substrates. The effects of ubiquitin epitope tags used to enrich ubiquitinated proteins remain incompletely understood, including whether purification biases exist and whether ubiquitin pathway enzymes utilize tagged and wild-type ubiquitin with equal efficiency.
Unfortunately, not many ubiquitination sites have currently been identified in mammalian cells. Therefore, information on how to manipulate ubiquitination and modulate some of the processes involving ubiquitination is lacking. Furthermore, methods to profile ubiquitination in cells and tissues are insufficient, and would require additional tools that allow for the simple, sensitive, specific, and rapid detection of ubiquitination sites in biological samples.
It is an object of the present invention to provide antibodies and a detection strategy to enable the sensitive, specific, and rapid detection of ubiquitination sites in biological samples.